Dynamin-Related Protein 1 Is Involved in Mitochondrial Damage, Defective Mitophagy, and NLRP3 Inflammasome Activation Induced by MSU Crystals

Figure 7

Drp1 knockdown relieved MSU crystal-induced NLRP3 inflammasome activation. (a)–(d) BMDMs were transfected with Drp1 siRNA or Ctrl siRNA, primed with LPS (100 ng/ml, 1 h), and challenged with MSU suspension (75 μg/ml, 12 h). (a) Secretion of IL-1β was detected by ELISA in the culture supernatants. (b) NLRP3, caspase-1 (pro form and cleaved p20), and IL-1β (pro form) expression was assessed by western blotting. Mature IL-1β and the p20 fragment of caspase-1 were also detected in cell culture supernatants. Tubulin was used as the loading control. (c) Total cell lysates were obtained in Triton X-100–containing buffer. Insoluble (pellet) fractions were crosslinked with DSS to capture ASC oligomers. The soluble and insoluble fractions were analyzed by immunoblotting with an ASC antibody (Ab). GAPDH was used as a loading control. Blots are representative of 3 independent experiments. (d) Representative LCM images of BMDMs stain with ASC Ab. DAPI stains nuclei. Arrows indicate ASC specks. Scale bars, 10 μm. Results are (). vs. without MSU crystal treatment; ^# vs. MSU crystal treatment + vehicle.

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